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Alphavirus Encephalitides

Chapter 12

ALPHAVIRUS ENCEPHALITIDES

KEITH E. STEELE, DVM, P h D * ; DOUGLAS S. REED, P h D † ; PAMELA J. GLASS, P h D ‡ ; MARY KATE HART, P h D § ; GEORGE

V. LUDWIG, P h D ¥ ; WILLIAM D. PRATT, DVM, P h D ¶ ; MICHAEL D. PARKER, P h D ** ; and JONATHAN F. SMITH, P h D ††

INTRODUCTION

HISTORY AND SIGNIFICANCE

ANTIGENICITY AND EPIDEMIOLOGY

Antigenic and Genetic Relationships

Epidemiology and Ecology

STRUCTURE AND REPLICATION OF ALPHAVIRUSES

Virion Structure

PATHOGENESIS

CLINICAL DISEASE AND DIAGNOSIS

Venezuelan Equine Encephalitis

Eastern Equine Encephalitis

Western Equine Encephalitis

Differential Diagnosis of Alphavirus Encephalitis

Medical Management and Prevention

IMMUNOPROPHYLAXIS

Relevant Immune Effector Mechanisms

Passive Immunization

Active Immunization

SUMMARY

* Colonel, US Army; Director, Division of Pathology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland 21702

† Microbiologist, Center for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland 21702

‡ Microbiologist, Division of Virology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland 21702

§ Director, Nonclinical Research, Dynport Vaccine Company, 64 Thomas Johnson Drive, Frederick, Maryland 21702; formerly, Chief, Division of Virol-

ogy, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

¥ Deputy Principal Assistant for Research and Technology, US Army Medical Research and Materiel Command, 504 Scott Street, Suite 204, Fort

Detrick, Maryland 21702; formerly, Science Director, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland

¶ Microbiologist, Division of Viral Biology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

21702

** Chief, Viral Biology Branch, Division of Virology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick,

Maryland 21702

†† Chief Scientific Officer, Alphavax, Incorporated, 2 Triangle Drive, Research Triangle Park, North Carolina 27709; formerly, Chief, Division of Viral

Biology, US Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Maryland

241

Medical Aspects of Biological Warfare

INTRODUCTION

During the 1930s, three distinct but antigenically

related viruses recovered from moribund horses were

shown to be previously unrecognized agents of se-

vere equine encephalitis. Western equine encephalitis

(WEE) virus was isolated in the San Joaquin Valley in

California in 1930 1 ; eastern equine encephalitis (EEE)

virus was isolated in Virginia and New Jersey in 1933 2,3 ;

and Venezuelan equine encephalitis (VEE) virus was

isolated in the Guajira Peninsula of Venezuela in

1938. 4 By 1938 it was clear that EEE and WEE viruses

were also natural causes of encephalitis in humans. 5-7

Naturally acquired human infections with VEE virus

occurred in Colombia in 1952 in association with an

equine epizootic. 8

Although these viruses cause similar clinical syn-

dromes in horses, the consequences of the infections

they cause in humans differ. EEE is the most severe of

the arboviral encephalitides, with case fatality rates of

50% to 70%, and neurological sequelae are common in

survivors. WEE virus appears to be less neuroinvasive

but has a pathology similar to that of EEE in patients

with encephalitis. In contrast, severe encephalitis result-

ing from VEE virus is rare in humans except for children.

In adults, the VEE virus usually causes an acute, febrile,

incapacitating disease with prolonged convalescence.

The three viruses are members of the Alphavirus

genus of the family Togaviridae . As with most of the

alphaviruses, VEE, EEE, and WEE are transmitted by

mosquitoes and maintained in cycles with various

vertebrate hosts. Environmental factors that affect the

interactions of the relevant mosquito and reservoir

host populations control the natural epidemiology

of these viruses. Of the 32 viruses classified within

this group, VEE, EEE, and WEE are the only viruses

regularly associated with encephalitis. Although these

encephalitic strains are restricted to the Americas, as a

group, alphaviruses have worldwide distribution and

include other epidemic human pathogens. Among

those pathogens, chikungunya virus (Asia and Africa),

Mayaro virus (South America), O’nyong-nyong virus

(Africa), Ross River virus (Australia), and Sindbis

virus (Africa, Europe, and Asia) can cause an acute

febrile syndrome often associated with debilitating

polyarthritic symptoms.

Although natural infections with the encephalitic

alphaviruses are acquired by mosquito bite, these vi-

ruses are also highly infectious by aerosol. VEE virus

has caused more laboratory-acquired disease than any

other arbovirus. Since its initial isolation, at least 150

symptomatic laboratory infections have been reported,

most of which were known or thought to be aerosol

infections. 9 Before vaccines were developed, most

laboratories working with VEE virus reported dis-

ease among their personnel. In one incident reported

in 1959 at the Ivanovskii Institute in Moscow, in the

former Soviet Union, at least 20 individuals developed

disease within 28 to 33 hours after a small number of

vials containing lyophilized virus were dropped and

broken in a stairwell. 10,11 The ability of aerosolized

EEE and WEE viruses to infect humans is less certain,

although the possibility is implied from animal studies.

Additionally, WEE viruses are less commonly studied

in the laboratory than VEE virus, and fewer human

exposures may explain the lower incidence of labora-

tory-acquired infections.

Perhaps as a consequence of their adaptation to

dissimilar hosts in nature, the alphaviruses replicate

readily and generally to high titers, in a wide range

of cell types and culture conditions. Virus titers of

1 billion infectious units per milliliter are not unusual,

and the viruses are stable in storage and in a variety

of laboratory procedures. Because they can be easily

manipulated in the laboratory, these viruses have long

served as model systems to study various aspects of

viral replication, pathogenesis, induction of immune

responses, and virus–vector relationships. As a result,

the alphaviruses are well described, and their charac-

teristics are well defined. 12,13

The designers of offensive biological warfare

programs initiated before or during World War II 14

recognized that the collective in-vitro and in-vivo

characteristics of alphaviruses, especially the equine

encephalomyelitis viruses, lend themselves well to

weaponization. Although other encephalitic viruses

could be considered as potential weapons (eg, the

tick-borne encephalitis viruses), few possess as many

of the required characteristics for strategic or tactical

weapon development as the alphaviruses:

• These viruses can be produced in large

amounts in inexpensive and unsophisticated

systems.

• They are relatively stable and highly infectious

for humans as aerosols.

• Strains are available that produce either inca-

pacitating or lethal infections.

• The existence of multiple serotypes of VEE and

EEE viruses, as well as the inherent difficul-

ties of inducing efficient mucosal immunity,

confound defensive vaccine development.

The equine encephalomyelitis viruses remain as

highly credible threats, and intentional release as a

small-particle aerosol, from a single airplane, could

242

Alphavirus Encephalitides

be expected to infect a high percentage of individuals

within an area of at least 10,000 km 2 . Furthermore,

these viruses are readily amenable to genetic manipu-

lation by modern recombinant DNA technology. This

characteristic is being used to develop safer and more

effective vaccines, 15,16 yet, in theory, it could also be

used to increase the weaponization potential of equine

encephalomyelitis viruses.

HISTORY AND SIGNIFICANCE

Descriptions of encephalitis epizootics in horses

thought to have been caused by EEE virus were re-

corded as early as 1831 in Massachusetts. 17 However,

it was not until the outbreaks of EEE in Delaware,

Maryland, and Virginia in 1933 and 1934 that the virus

was isolated. During a similar outbreak in North Caro-

lina in 1935, birds were first suspected as the natural

reservoir. 18 The initial isolation of EEE virus from a

bird 19 and from Culiseta melanura mosquitoes, 20 the

two major components of the EEE natural cycle, were

both reported in 1951. Outbreaks of EEE virus have

occurred in most eastern states and in southeastern

Canada but have been concentrated along the east-

ern and Gulf coasts. Although only 211 EEE cases in

humans were reported 21 between 1938 and 1985, the

social and economic impact of this disease has been

larger than might be expected because of the high

fatality rate, equine losses, extreme concern among

individuals living in endemic areas during outbreaks,

and the surveillance and mosquito-control measures

required. Isolation of EEE virus from Aedes albopictus

mosquitoes, which were recently introduced into EEE

endemic areas in the United States, has heightened

concern because of the opportunistic feeding behavior

of these mosquitoes and their apparent high vector

competence for EEE virus. 22

The initial isolation in 1930 of WEE virus from the

brain tissues of a horse with encephalitis was made

during a large and apparently unprecedented epizootic

in California, which involved at least 6,000 horses with

an approximate mortality of 50%. 1 Cases of human

encephalitis in California were not linked to WEE until

1938, when the virus was isolated from the brain of a

child. During the 1930s and 1940s, several other exten-

sive epizootics occurred in western and north-central

states, as well as Saskatchewan and Manitoba in Cana-

da, and affected large numbers of equines and humans.

For example, it has been estimated that during 1937

and 1938, more than 300,000 equines were infected in

the United States, and in Saskatchewan, 52,500 horse

infections resulted in 15,000 deaths. 23,24 Unusually high

numbers of human cases were reported in 1941: 1,094

in Canada and 2,242 in the United States. The attack

rate in these epidemics ranged from 22.9 to 171.5 per

100,000, with case fatality rates of 8% to 15%. 24

In the early 1940s, workers isolated WEE virus

from Culex tarsalis mosquitoes 25 and demonstrated the

presence of specific antibody to WEE virus in birds, 26

suggesting that birds are the reservoirs of the virus

in nature. The annual incidence of disease in both

equines and humans continues to vary widely, which

is indicative of an arthropod-borne disease. Significant

epidemics occurred in 1952, 1958, 1965, and 1975. 24

VEE virus was initially isolated during investiga-

tions of an epizootic occurring in horses in Venezuela

in 1936, and the isolate was shown to be antigenically

different from the EEE and WEE viruses isolated pre-

viously in the United States. 4,27 Over the following

30 years, many VEE outbreaks were reported among

horses, and humans became infected in large numbers

in association with these epizootics. 28 Most of those

infected recovered after suffering an acute, febrile

episode, but severe disease with encephalitis and death

also occurred, mostly in children and older individu-

als. Major epizootics occurred in Venezuela, Colombia,

Peru, and Ecuador in the 1960s, apparently spreading

to Central America in 1969. 29 These epizootics and

previous ones were associated with costly and dire

consequences, especially among rural people, who

not only had the disease but also lost their equines,

which were essential for transportation and agricul-

ture. Between 1969 and 1971, epizootics were reported

in essentially all of Central America and subsequently

continued north to Mexico and into Texas. The most

recent major epizootic occurred in Venezuela and

Colombia in 1995. 30

Between active epizootics, it was not possible to

isolate the equine virulent viruses. During the 1950s

and 1960s, however, several other attenuated, antigeni-

cally different VEE strains were isolated from different

geographical areas. These enzootic strains could be

differentiated antigenically not only among themselves

but also from the epizootic strains. 31 Enzootic strains

used different mosquito vectors than the epizootic

strains 32 and used rodents as reservoir hosts. 33 Many of

the enzootic strains, however, proved equally patho-

genic for humans.

Therefore, within 30 years of the initial isolation

of the EEE, WEE, and VEE viruses, an accurate pic-

ture had emerged of their endemic and epidemic

behavior, arthropod vectors, reservoir hosts, and the

diseases produced. Although not yet understood at

the molecular level, these three viruses were well

described as agents of disease, and the basic methods

243

Medical Aspects of Biological Warfare

for their manipulation and production were known.

The development of this knowledge occurred dur-

ing the same period of war and political instability

that fostered the establishment of biological warfare

programs in the United States 34 and elsewhere, and it

was evident that the equine encephalomyelitis viruses

were preeminent candidates for weaponization. The

viruses were incorporated into these programs for

both potential offensive and defensive reasons. The

offensive biological warfare program in the United

States was disestablished in 1969, and all stockpiles

were destroyed 14 by executive order, which stated:

warfare. The United States shall conine its biological re-

search to defensive measures such as immunization and

safety measures . 35

Continuing efforts within the defensive program

in the 1960s and 1970s produced four vaccines for the

encephalomyelitis viruses: live attenuated (TC-83)

and formalin-inactivated (C84) vaccines for VEE, and

formalin-inactivated vaccines for EEE and WEE. These

vaccines are used under investigational new drug

status for at-risk individuals, distributed under in-

vestigational new drug provisions, and recommended

for use by any laboratory working with these viruses. 9

Although these vaccines are useful, they have certain

disadvantages (discussed later in this chapter), and

second-generation vaccines are being developed. 15

The United States shall renounce the use of lethal biological

agents and weapons and all other methods of biological

ANTIGENICITY AND EPIDEMIOLOGY

Antigenic and Genetic Relationships

mosquitoes of the Melanoconion subgenus. 47-49 Infection

of equines with some enzootic subtypes leads to an

immune response capable of protecting the animals

from challenge with epizootic strains. 50 Limited data,

acquired following laboratory exposures, suggest that

cross-protection between epizootic and enzootic strains

may be much less pronounced in humans. 51-53

The three American equine encephalitides virus

complexes, VEE, EEE, and WEE, have been grouped

with four additional virus complexes into the Alpha-

virus genus based on their serologic cross-reactivity

(Table 12-1). 13 Analysis of structural gene sequences

obtained from members of the VEE and EEE virus com-

plexes confirms the antigenic classification and serves

as another tool for classifying these viruses (Figure 12-

1). The WEE virus complex, including Highlands J, Fort

Morgan, and WEE viruses, is identified as recombinant

viruses originating from ancestral precursors of EEE

and Sindbis viruses and, therefore, falls into a unique

genetic grouping of alphaviruses. 36-39

Eastern Equine Encephalitis Virus Complex

The EEE virus complex consists of viruses in two

antigenically distinct forms: (1) the North American

and (2) the South American variants. 54 The two forms

can be distinguished readily by hemagglutination in-

hibition and plaque-reduction neutralization tests. 54,55

All North American and Caribbean isolates show a

high degree of genetic and antigenic homogeneity.

However, they are distinct from the South American

and Central American isolates, which tend to be more

heterogeneous and form three genetic clades that are

readily distinguished from the monophyletic North

American EEE viruses. 56,57

EEE is endemic to focal habitats ranging from south-

ern Canada to northern South America. The virus has

been isolated as far west as Michigan but is most com-

mon along the eastern coast of the United States between

New England and Florida. Enzootic transmission of EEE

virus occurs almost exclusively between passerine birds

(eg, the perching songbirds) and the mosquito Culiseta

melanura . Because of the strict ornithophilic feeding

behavior of this mosquito, human and equine disease re-

quires the involvement of more general feeders, known

as bridging vectors, such as members of the genera Aedes

and Coquilletidia . Mosquito vectors belonging to Culex

species may play a role in maintaining and transmitting

South American EEE strains. 58

Venezuelan Equine Encephalitis Virus Complex

The VEE virus complex consists of six closely re-

lated subtypes that manifest different characteristics

with respect to ecology, epidemiology, and virulence

for humans and equines (Table 12-2). The IA/B and C

varieties are commonly referred to as epizootic strains.

These strains, which have been responsible for exten-

sive epidemics in North, Central, and South America,

are highly pathogenic for humans and equines. All

epizootic strains are exotic to the United States and

have been isolated from areas where virus occurs

naturally. 40 Subtypes II, III, IV, V, and VI and varieties

ID, IE, and IF are referred to as the enzootic strains. 41-46

Like the epizootic strains, the enzootic strains may

cause disease in humans, but they differ from the

epizootic strains in their lack of virulence for equines.

The enzootic viruses are commonly isolated in specific

ecological habitats, where they circulate in transmis-

sion cycles primarily involving rodents and Culex

244

Alphavirus Encephalitides

TABLE 12-1

ANTIGENIC CLASSIFICATION OF ALPHAVIRUSES

Virus

Antigenic Complex

Species

Subtype

Variety

Western Equine Encephalitis (WEE)

WEE

Y 62-33

Highlands J

Fort Morgan

Aura

Sindbis

Ockelbo

Babanki

Whataroa

Kyzylagach

Venezuelan Equine Encephalitis (VEE)

VEE

A-B

II Everglades

III Mucambo

Mucambo

Tonate

71D-1252

IV Pixuna

V Cabassou

VI AG80-663

Eastern Equine Encephalitis (EEE)

EEE

North American

South American

Semliki Forest

Chikungunya

Several

O’nyong-nyong

Igbo ora

Getah

Sagiyama

Ross River

Mayaro

Una

Middelburg

Nduma

Barmah Forest

Adapted with permission from Peters CJ, Dalrymple JM. Alphaviruses. In: Fields BM, Knipe DM, eds. Virology . 3rd ed, Vol 1. New York,

NY: Raven Press; 1990: 716.

Western Equine Encephalitis Virus Complex

tests. In addition, WEE complex viruses isolated in the

western United States (ie, WEE) are antigenically and

genetically distinct from those commonly found in the

eastern United States (ie, Highlands J). 57,59 Sindbis virus

is considered a member of the WEE virus complex

based on antigenic relationships. However, sequence

comparisons show that WEE, Highlands J, and Fort

Morgan viruses are actually derived from a recom-

bination event between ancestral Sindbis and EEE

viruses. The structural domains of the recombinant

viruses were derived from the Sindbis virus ancestor,

Six viruses, WEE, Sindbis, Y 62-63, Aura, Fort Mor-

gan, and Highlands J, comprise the WEE complex.

Several antigenic subtypes of WEE virus have been

identified, but their geographical distributions over-

lap. 40 Most of the members of the WEE complex are

distributed throughout the Americas, but subtypes

of Sindbis virus and its subtypes have strictly Old

World distributions. 13 The New World WEE complex

viruses can be distinguished readily by neutralization

245

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